Method for purifying cannabinoids

ABSTRACT

The invention relates to a method for the chromatographic purification of at least one cannabinoid compound, wherein the method comprises a main purification stage comprising the steps of: injecting an initial mixture comprising the at least one cannabinoid compound and one or more additional compounds onto a main stationary phase comprising silica particles, the silica particles comprising amino and/or diol groups; performing an elution with an elution solution, and collecting one or more elution fractions; and optionally, washing the main stationary phase with a washing solution and collecting one or more washing fractions; at least one of the elution fractions or washing fractions containing the at least one cannabinoid compound purified from the one or more additional compounds.

TECHNICAL FIELD

The present invention relates to a method for purifying cannabinoids,the method comprising a step of putting an initial mixture containing atleast one cannabinoid in contact with a silica-based stationary phasecomprising amino and/or diol groups.

TECHNICAL BACKGROUND

For a long time, Cannabis has been considered to have medicinalproperties for the treatment of various diseases and disorders such ascramps, migraines, convulsions, and attenuation of nausea and vomiting.Therefore, Cannabis can be used for example to stimulate the appetite ofa person suffering from HIV infection, to treat nausea and vomitingprovoked by chemotherapy, or to reduce muscle cramps and spasms inpatients suffering from multiple sclerosis.

Cannabinoids are the major active constituents of the plant Cannabissativa. The most important natural cannabinoids present in herbalCannabis are Δ⁹-tetrahydrocannabinolic acid (Δ⁹-THCA) and cannabidiolicacid (CBDA) with small amounts of the corresponding neutral cannabinoidsΔ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD). While Δ⁹-THC ispsychoactive, CBD is a pharmaceutically active compound devoid ofpsychoactive activity. Other important cannabinoids are cannabigerol(CBG), cannabinol (CBN), cannabinidiol (CBND) cannabichromene (CBC) andΔ⁸-tetrahydrocannabinol (Δ⁸-THC).

Although, in some cases, patients may use crude marijuana to reduce ortreat their symptoms, this product is less suitable for pharmaceuticalapplications. It is therefore preferable to separate and purify thecannabinoids present in herbal Cannabis in order to use them inpharmaceutical formulations.

In addition, due to the number and amount of phytosanitary substancesused for the cultivation of Cannabis, it is important to eliminate everytrace of phytosanitary substances from the cannabinoid compounds so thatthey can be used in pharmaceutical formulations.

Similar needs arise in the context of the development ofCannabis-derived compositions for recreational use.

Document U.S. Pat. No. 8,895,078 relates to a method for producing anextract from Cannabis plant matter, containing tetrahydrocannabinol,cannabidiol and optionally their corresponding carboxylic acids. Thedocument also relates to the production of cannabidiol from the extract.

Documents U.S. Pat. No. 7,700,368 and GB 2 393 182 disclose methods forthe preparation of cannabinoids in substantially pure forms startingfrom plant materials.

Document U.S. Pat. No. 6,403,126 describes a method for extractingcannabinoids, cannflavins and/or essential oils from hemp. The documentalso describes a method for the production of a hemp extract lackingΔ⁹-THC.

Document WO 2013/165251 relates to a method for preparing a Δ⁹-THCisolate from a crude solvent extract of Cannabis plant material.

Document U.S. Pat. No. 6,365,416 describes a method for preparing THC byextraction of a plant material with a non-polar solvent followed byvacuum distillation and chromatography.

There is still a need for an improved method for purifying cannabinoidcompounds, notably allowing separation between cannabinoid compounds andimpurities such as phytosanitary substances, as well as allowingseparation between the different cannabinoid compounds themselves.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for thechromatographic purification of at least one cannabinoid compound,wherein the method comprises a main purification stage comprising thesteps of:

-   -   injecting an initial mixture comprising the at least one        cannabinoid compound and one or more additional compounds onto a        main stationary phase comprising silica particles, the silica        particles comprising amino and/or diol groups;    -   performing an elution with an elution solution, and collecting        one or more elution fractions; and    -   optionally, washing the main stationary phase with a washing        solution and collecting one or more washing fractions;

at least one of the elution fractions or washing fractions containingthe at least one cannabinoid compound purified from the one or moreadditional compounds.

According to some embodiments, this method further comprises apreliminary purification stage before the main purification stage, saidpreliminary purification stage comprising the steps of:

-   -   putting a preliminary mixture comprising the at least one        cannabinoid compound, the one or more additional compounds and        one or more further compounds in contact with a preliminary        stationary phase; and    -   collecting at least one liquid fraction enriched in the at least        one cannabinoid compound and the one or more additional        compounds and depleted in the further compounds, said at least        one liquid fraction providing the initial mixture.

According to some embodiments, the preliminary stationary phasecomprises silica particles comprising amino and/or diol groups.

According to some embodiments, the preliminary purification stagecomprises putting the preliminary mixture in contact with thepreliminary stationary phase in a suspension solution, to form asuspension; filtering so as to collect a first fraction; optionally,resuspending the preliminary stationary phase in one or more furthersolutions, and filtering so as to collect one or more further fractions;the liquid fraction enriched in the at least one cannabinoid compoundand the one or more additional compounds and depleted in the furthercompounds being at least one of the first and further fractions.

According to some embodiments, the initial mixture is a Cannabisextract.

According to some embodiments, the at least one cannabinoid compound isselected from Δ⁹-tetrahydrocannabinol, cannabidiol, cannabinol,cannabigerol, cannabichromene, cannabidivarol, tetrahydrocannabidiol,tetrahydrocannabidivarol, Δ⁸-tetrahydrocannabinol, carboxylic acidprecursors of the foregoing compounds, related naturally occurringcompounds and their derivatives, and combinations thereof.

According to some embodiments, the one or more additional compounds areselected from cannabinoid compounds, pesticides, plant growth regulatorsand combinations thereof.

According to some embodiments, the one or more further compounds areselected from cannabinoid compounds, pesticides, plant growth regulatorsand combinations thereof.

According to some embodiments, each of the elution solution and thewashing solution is a mixture of a polar solvent and a non-polarsolvent; the polar solvent independently being selected from ethanol,methanol and water, and the non-polar solvent being independentlyselected from pentane, hexane, heptane and octane.

According to some embodiments, each of the elution solution and thewashing solution is a mixture of a polar solvent and a non-polarsolvent; the elution solution having a volume ratio of non-polar solventto polar solvent from 99/1 to 50/50; and the washing solution having avolume ratio of non-polar solvent to polar solvent from 1/99 to 50/50.

According to some embodiments, the method further comprisesequilibrating the main stationary phase with an equilibration solutionin order to equilibrate the main stationary phase for a furtherinjection, the elution solution and the equilibration solution being thesame.

According to some embodiments, the method is carried out in aninstallation chosen from a single column installation, or a multi-columninstallation having a total bed length smaller than 100 cm.

According to some embodiments, the preliminary purification stage iscarried out in an installation chosen from a single column installation,or a multi-column installation having a total bed length smaller than100 cm, and the main purification stage is carried out in a multi-columninstallation having a total bed length smaller than 100 cm.

According to some embodiments, the method is carried out in aninstallation comprising one or more chromatographic columns having adiameter equal to or larger than 5 cm.

According to some embodiments, the installation has a total dead volumelower than or equal to 30% of the total volume of main stationary phasein the installation.

According to some embodiments, the injection of the initial mixture isperformed continuously.

According to some embodiments, the injection of the initial mixture isperformed in a discontinuous way.

According to some embodiments, the weight proportion of the purified atleast one cannabinoid compound relative to the amount of the at leastone cannabinoid compound present in the initial mixture is at least 95%.

According to some embodiments, the at least one elution fraction orwashing fraction containing the at least one cannabinoid compoundpurified from the one or more additional compounds further comprises aweight proportion of the one or more additional compounds which is lessthan 5%, relative to the amount of the one or more additional compoundspresent in the initial mixture.

According to some embodiments, the preliminary purification stagecomprises putting in contact the preliminary mixture with thepreliminary stationary phase by injecting the preliminary mixture ontothe preliminary stationary phase; performing an elution with an elutionsolution, and collecting one or more preliminary elution fractions; andoptionally washing the preliminary stationary phase with a washingsolution and collecting one or more preliminary washing fractions; atleast one of said preliminary elution fractions and/or preliminarywashing fractions being the liquid fraction enriched in the at least onecannabinoid compound and the one or more additional compounds anddepleted in the further compounds.

The present invention makes it possible to address the need mentionedabove. In particular the invention provides an improved method forpurifying cannabinoid compounds, notably allowing separation betweencannabinoid compounds and impurities such as phytosanitary substances aswell as allowing separation between the different cannabinoid compoundsthemselves.

This is achieved by using silica particles which comprise amino and/ordiol groups. As a result, the polarity of the silica is increasedcompared to bare silica, therefore improving separation not only betweenthe cannabinoid compounds and impurities such as phytosanitarysubstances but also between different cannabinoid compounds present inthe same initial mixture.

In some embodiments, the invention makes it possible to purifycannabinoid compounds in a more efficient manner, i.e. with increasedyield and/or with increased selectivity and/or within a shorter timeand/or with a lower consumption of solvent.

In the prior art of chromatography, some expressions may be used withdifferent meanings. For the sake of clarity, the following definition ofthe expressions “elution solution” and “washing solution” is presented.Different components of a feed have to be considered, depending on theirretention times, compared with the retention time of a non-retainedproduct. These components may be considered as:

-   -   weakly retained when their retention times are lower than twice        the retention time of a non-retained product; these weakly        retained components are collected with the use of an elution        solution;    -   highly retained when their retention times are from 2 to        approximately 15 times the retention time of a non-retained        product; these highly retained components are collected with the        use of an elution solution and may require a modulation of the        eluent strength;    -   strongly retained or fixed when their retention times are higher        than approximately 15 times the retention time of a non-retained        product; these strongly retained components are removed and        collected with the use of a washing solution.

An elution solution is thus a solution that can elute weakly and highlyretained products. The eluent strength of the elution solution can beincreased to decrease the retention times of some components.

A washing solution is a solution with a high elution strength that canremove strongly retained or fixed components from the stationary phase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 5 are chromatograms obtained when implementing cannabinoidpurification according to various embodiments of the invention, asdescribed in further detail in the example section. The time (inminutes) is shown on the x-axis and the intensity of the signal is shownon the y-axis.

DESCRIPTION OF EMBODIMENTS

The invention will now be described in more detail without limitation inthe following description.

The method of the invention comprises a main purification stage, whereinan initial mixture comprising at least one cannabinoid compound and oneor more additional compounds (such as impurities or contaminants) issubjected to chromatographic separation using a main stationary phasecomprising silica particles comprising amino and/or diol groups.

Optionally, the method of the invention may also comprise a preliminarypurification stage, before the main purification stage, wherein theinitial mixture is obtained, starting from a preliminary mixturecontaining the at least one cannabinoid compound, the one or moreadditional compounds as well as one or more further compounds.

The starting material of the method of the invention is thus either theinitial mixture or the preliminary mixture, depending on whether thepreliminary purification stage is present or not.

The at least one cannabinoid compound recovered in a purified form maybe used in particular in a therapeutic composition, or in a recreationalcomposition. It can in particular be formulated as an electroniccigarette composition.

Starting Material

Aside from the at least one cannabinoid compound, the one or moreadditional compounds, and optionally the one or more further compounds,the starting material may notably comprise water or may be a water-basedsolution. Alternatively, the starting material may comprise or be basedon a non-aqueous solvent. Alternatively, the starting material may be inthe form of a dry extract.

The starting material may comprise one or more cannabinoid compounds forexample derived from a Cannabis plant. The term “Cannabis plant” refersto wild type Cannabis sativa and also variants thereof, includingCannabis chemovars which naturally contain different amounts ofindividual cannabinoids, including also Cannabis sativa subspeciesindica including the variants var. indica and var. kafiristanica,Cannabis indica as well as plants which are the result of geneticcrosses, self-crosses or hybrids thereof.

The starting material may comprise at least one cannabinoid compoundselected from Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD),cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC),cannabidivarol (CBDV), tetrahydrocannabidiol (THCBD),tetrahydrocannabidivarol (THCBDV), tetrahydrocannabigerol (THCBG),tetrahydrocannabichromene (THCBC), Δ⁸-tetrahydrocannabinol, carboxylicacid precursors of the foregoing compounds, related naturally occurringcompounds and their derivatives.

According to some embodiments the starting material comprises a singlecannabinoid compound.

According to some preferred embodiments, the starting material comprisesmore than one cannabinoid compound. In this case, at least one orpossibly several of the cannabinoids are sought to be purified. Othersof the cannabinoid compounds may optionally be considered as impuritiesor contaminants (herein, “additional compounds” and/or “furthercompounds”) and sought to be separated and removed.

Preferably, the starting material comprises at least Δ⁹-THC and/or CBDand/or CBN.

According to some embodiments, the starting material may comprise from 5to 100% of cannabinoid compounds, and preferably from 10 to 100% ofcannabinoid compounds, based on the total dry weight of the startingmaterial. The starting material may therefore comprise from 5 to 10%; orfrom 10 to 20%; or from 20 to 30%; or from 30 to 40%; or from 40 to 50%;or from 50 to 60%; or from 60 to 70%; or from 70 to 80%; or from 80 to90%; or from 90 to 100% of cannabinoid compounds, based on the total dryweight of the starting material.

When the starting material comprises Δ⁹-THC, the Δ⁹-THC can representfrom 0.01 to 99.9%, and preferably from 0.1 to 95% of the total dryweight of the starting material. For example, Δ⁹-THC can represent from0.01 to 0.1%; or from 0.1 to 0.5%; or from 0.5 to 1%; or from 1 to 2%;or from 2 to 3%; or from 3 to 4%; or from 4 to 5%; or from 5 to 10%; orfrom 10 to 15%; or from 15 to 20%; or from 20 to 25%; or from 25 to 30%;or from 30 to 35%; or from 35 to 40%; or from 40 to 45%; or from 45 to50%; or from 50 to 55%; or from 55 to 60%; or from 60 to 65%; or from 65to 70%; or from 70 to 75%; or from 75 to 80%; or from 80 to 85%; or from85 to 90%; or from 90 to 95%; from 95 to 99.9% of the total dry weightof the starting material.

When the starting material comprises CBD, the CBD can represent from0.01 to 9.9%, and preferably from 0.1 to 95% of the total dry weight ofthe starting material. For example, CBD can represent from 0.01 to 0.1%;or from 0.1 to 0.5%; or from 0.5 to 1%; or from 1 to 2%; or from 2 to3%; or from 3 to 4%; or from 4 to 5%; or from 5 to 10%; or from 10 to15%; or from 15 to 20%; or from 20 to 25%; or from 25 to 30%; or from 30to 35%; or from 35 to 40%; or from 40 to 45%; or from 45 to 50%; or from50 to 55%; or from 55 to 60%; or from 60 to 65%; or from 65 to 70%; orfrom 70 to 75%; or from 75 to 80%; or from 80 to 85%; or from 85 to 90%;or from 90 to 95%; from 95 to 99.9% of the total dry weight of thestarting material.

The starting material comprises one or more additional compounds andoptionally further compounds, to be separated from the at least onecannabinoid compound.

In some variations, the additional compounds and/or further compoundsmay comprise or consist of cannabinoid compounds, which may inparticular selected from the above list.

For example, the compound to be purified may be CBD, and the additionalcompounds and/or further compounds, to be separated from it, maycomprise or consist of Δ⁹-THC and/or CBN. Or the compound to be purifiedmay be CBN, and the additional compounds and/or further compounds, to beseparated from it, may comprise or consist of Δ⁹-THC and/or CBD. Or thecompound to be purified may be Δ⁹-THC, and the additional compoundsand/or further compounds, to be separated from it, may comprise orconsist of CBD and/or CBN.

In some variations, the additional compounds and/or further compoundsmay comprise or consist of pesticides and/or plant growth regulators.

Pesticides are substances intended to repel, kill or control any speciesdesignated as “pest”, including weed, insects, rodents, fungi or otherorganisms. Pesticides include herbicides, insecticides, rodenticides,fungicides and bactericides.

Plant growth regulators are substances or mixture of substances intendedfor accelerating or retarding the rate of growth or rate of maturation,or for otherwise altering the behavior of plants or the produce thereof.Plant nutrients, trace elements, nutritional chemicals, plant inoculantsand soil amendments are not considered as plant growth regulators.

Examples of pesticides and plant growth regulators include daminozide,neonicotinoids such as e.g. imidacloprid and thiamethoxam, strobilurinssuch as e.g. azoxystrobin and trifloxystrobin, triazoles such asmyclobutanil, avermectin, etoxazole, bifenazate as well as theirderivatives.

According to some embodiments, the starting material comprises onesingle additional compound which is to be separated from the at leastone cannabinoid compound.

According to other embodiments, the starting material comprises severaladditional compounds and/or further compounds which are to be separatedfrom the at least one cannabinoid compound.

In some embodiments, the additional compounds and/or further compoundscomprise or consist of one or more cannabinoid compounds on the onehand, plus one or more pesticides and/or plant growth regulators on theother hand.

According to some embodiments, the starting material may comprise from0.01 to 50% of the one or more additional compounds and/or furthercompounds, and preferably from 0.1 to 50% of the one or more additionalcompounds and/or further compounds, based on the total dry weight of thestarting material. For example, the starting material may comprise from0.01 to 0.1%; or from 0.1 to 0.5%; or from 0.5 to 1%; or from 1 to 2%;or from 2 to 3%; or from 3 to 4%; or from 4 to 5%; or from 5 to 10%; orfrom 10 to 15%; or from 15 to 20%; or from 20 to 25%; or from 25 to 30%;or from 30 to 35%; or from 35 to 40%; or from 40 to 45%; or from 45 to50% of the one or more additional compounds and/or further compounds.

In particular, the starting material may comprise from 0.01 to 50% ofone or more plant growth regulators and/or pesticides, and preferablyfrom 0.1 to 50% of one or more plant growth regulators and/orpesticides, based on the total dry weight of the starting material. Forexample, the starting material may comprise from 0.01 to 0.1%; or from0.1 to 0.5%; or from 0.5 to 1%; or from 1 to 2%; or from 2 to 3%; orfrom 3 to 4%; or from 4 to 5%; or from 5 to 10%; or from 10 to 15%; orfrom 15 to 20%; or from 20 to 25%; or from 25 to 30%; or from 30 to 35%;or from 35 to 40%; or from 40 to 45%; or from 45 to 50% of one or moreplant growth regulators and/or pesticides. One advantage of the presentinvention is that it makes it possible to separate cannabinoid compoundsfrom chemicals such as plant growth regulators and/or pesticides whichmay be present in minute amounts in a starting material.

Apart from the at least one cannabinoid compound to be purified and theone or more additional compounds and/or further compounds, the startingmaterial may also comprise natural waxes, terpenes and flavonoids.

The starting material may be an extract obtained from a Cannabis plant.The Cannabis plant may include different species including hemp andmarijuana. The starting material can be a Cannabis extract, notably ahemp extract, or a marijuana extract, or a mixture derived from one ofthose. By “marijuana extract” is meant an extract wherein Δ⁹-THC is themajor cannabinoid compound. By “hemp extract” is meant an extractenriched in CBD, and/or wherein CBD is the major cannabinoid compoundand wherein Δ⁹-THC is found in low levels.

The starting material may be obtained by extracting Cannabis flos with anon-polar solvent, an alcohol (such as ethanol) or liquid carbondioxide, preferably with a non-polar solvent. Any non-polar solventcapable of solubilizing the Cannabis plant can be used. Preferrednon-polar solvent include liquid non-polar solvents comprising C5 toC12, preferably C5 to C8 straight chain or branched chain alkanes. Morepreferably the non-polar solvent is hexane or heptane.

The starting material may be prepared by solubilizing a part of theCannabis plant in an extraction solvent, removing insoluble materialfrom the resulting solution and removing the extraction solvent from thesolution to form the initial mixture containing the at least onecannabinoid compound.

Main Stationary Phase

In the main purification stage, use is made of a main stationary phase,which comprises silica particles or preferably consists of silicaparticles.

These silica particles invention form a so-called silica gel when insolution. They comprise amino and/or diol groups on their surface. Inother words, the silica particles comprise grafted functions of aminesand/or diols. When used in a chromatographic installation, the silicaparticles form a normal phase, which means that the silica particles aremostly polar and separation between species is believed to mostly relyon polar interactions between the various species and the particles.

The silica particles may in particular comprise groups of formula (I) ontheir surface: (I) —Si—R—NH₂, wherein R is a linker group which may forinstance comprise from 1 to 20 carbon atoms, preferably from 1 to 10carbon atoms, more preferably from 1 to 4 carbon atoms, and optionallyat least one heteroatom, preferably oxygen. R may in particular be(CH₂—CH₂) or (CH₂—CH₂—CH₂). Alternatively, the silica particles maycomprise groups of formula (II) on their surface: (II)—Si—R₁(OH)—R₂(OH), wherein R₁ and R₂ are linker groups, each of whichmay for instance comprise from 1 to 20 carbon atoms, preferably from 1to 10 carbon atoms, more preferably from 1 to 6 carbon atoms, andoptionally at least one heteroatom, preferably oxygen. R₁ may inparticular be (CH₂)_(m)—O—(CH₂)_(n), wherein m and n are integerspreferably from 1 to 4, more preferably from 1 to 3, and for instancem=3 and n=1. R₂ may in particular be (CH₂)_(m) wherein m is an integerpreferably from 1 to 3, or from 1 to 2, and most preferably m is 1.

The silica particles may preferably have an average volume diameter(Dv50) equal to or less than 200 μm, or 150 μm, or 100 μm, or 50 μm, or20 μm.

In some embodiments, the Dv50 of the silica particles is from 1 μm to 50μm, preferably from 2 μm to 30 μm, or from 5 μm to 20 μm.

The term Dv50 refers to the 50th percentile of the particle sizedistribution, i.e. 50% of the particles have a size (corresponding tothe diameter of the particles, when the particles are spherical) lessthan the Dv50 and 50% have a size larger than the Dv50. This is themedian of the volumetric distribution of silica particles.

Preferably, the distribution of the volume diameter of the particles ofsilica has only one peak (monodispersed distribution). The uniformitycoefficient of this distribution may for example be less than or equalto 1.5, or less than or equal to 1.3, or less than or equal to 1.15. Theparticle size distribution of the silica particles may be determined bylaser granulometry (NF 13320 standard).

Main Purification Stage

In the main purification stage, the at least one cannabinoid compound issubstantially separated from the one or more additional compounds in achromatographic installation.

As a first step, the initial mixture is put into contact with the mainstationary phase comprising silica particles as described above, byinjecting the initial mixture onto said main stationary phase.

Before the initial mixture is put into contact with the main stationaryphase, it may optionally be diluted or dissolved (if it is a dryextract) in a solvent, or alternatively be used as such. Solvents whichcan be used for dissolving the initial mixture include C5-C12 alkanessuch as heptane and hexane, alcohols such as ethanol, or their mixtures.These solvents are preferably similar to the elution solution describedbelow but not limited to it.

Then, an elution step is performed by passing an elution solutionthrough the main stationary phase. Optionally, this is followed by awashing step which is performed by passing a washing solution throughthe main stationary phase.

At least one fraction is collected at the elution step.

If the washing step is carried out, at least one fraction is collectedat the washing step.

In some embodiments, a single fraction is collected at the elution step.In other embodiments, at least two fractions are collected at theelution step.

In some embodiments, a single fraction is collected at the washing step.In other embodiments, at least two fractions are collected at thewashing step.

More than one fraction may be collected at different points in time in agiven step.

At least one of the collected fractions contains the at least one(desired) cannabinoid compound purified from the one or more additionalcompounds, which means that the fraction in enriched in the at least onecannabinoid compound and depleted in the one or more additionalcompounds.

By a fraction “enriched” in one species A and “depleted” in one speciesB, it is meant that the ratio of species A/species B weightconcentrations in the fraction is greater than that of the initialmixture (independently of the effects of concentration or overalldilution).

The ratio may be greater by at least a factor of 10 or 100 or 1000 or10000.

The weight proportion of the purified at least one cannabinoid compoundrelative to the amount of the at least one cannabinoid compound presentin the initial mixture may be at least 95%, preferably at least 98%,more preferably at least 99%, and most preferably at least 99.5% or99.9%.

The weight proportion of the one or more additional compounds recoveredin the at least one elution fraction or washing fraction comprising thepurified at least one cannabinoid compound relative to the amountpresent in the initial mixture may be less than 5%, preferably at least2%, more preferably less than 1%, and most preferably less than 0.5% orless than 0.1%.

The weight proportion of the one or more additional compounds relativeto the purified at least one cannabinoid compound, in the at least oneelution fraction or washing fraction comprising the purified at leastone cannabinoid compound, may in particular be less than 2%, preferablyless than 1%, more preferably less than 0.5%, most preferably less than0.1% or less than 0.05% or less than 0.01%.

Other collected fractions may be enriched in one or several of the oneor more additional compounds and depleted in the at least one (desired)cannabinoid compound.

The weight proportion of the one or more additional compounds recoveredin fractions other than the fraction containing the purified at leastone cannabinoid compound, relative to the amount present in the initialmixture, may be at least 95%, preferably at least 98%, more preferablyat least 99%, and most preferably at least 99.5% or 99.9%.

In some embodiments, the fraction containing the purified at least onecannabinoid compound is obtained at the elution step.

In some embodiments, at least one fraction enriched in additionalcompound(s) may be obtained at the elution step.

In some embodiments, at least one fraction enriched in additionalcompound(s) may be obtained at the washing step.

In some embodiments, at least one fraction enriched in additionalcompound(s) may be obtained at the elution step and at least onefraction enriched in other additional compound(s) may be obtained at thewashing step.

In some embodiments, at least part, and possibly all of the additionalcompounds present in the initial mixture have a stronger affinity forthe main stationary phase than the at least one cannabinoid compound andare therefore retained on it, during the elution step (and optionallywashed during the washing step).

In some embodiments, at least part, and possibly all of the additionalcompounds have a weaker affinity for the main stationary phase than theat least one cannabinoid compound and are therefore eluted during theelution step before the at least one cannabinoid compound, and collectedseparately.

In some embodiments, some of the additional compounds have a weakeraffinity for the main stationary phase than the at least one cannabinoidcompound and others of the additional compounds have a stronger affinityfor the main stationary phase than the at least one cannabinoidcompound. These respective additional compounds are thus collected indifferent fractions.

In some exemplary embodiments, during the elution step, one firstfraction containing a mixture of cannabinoid compounds (including forexample Δ⁹-THC, CBD and CBN) is recovered, as well as one or more otherfractions containing additional compounds (such as pesticides or plantgrowth regulators) which may for instance be more polar than thecannabinoid compounds. The first fraction is substantially free fromthese additional compounds. Neonicotinoids (such as thiamethoxam andimidacloprid) are examples of such additional compounds. Otheradditional compounds may be coeluted in the first fraction, and may beseparated at another stage. Yet other additional compounds may remainbound to the main stationary phase and may be released and collected ina fraction during a subsequent washing step.

In other exemplary embodiments, during the elution step, two or morefractions containing cannabinoid compounds may be recovered, andoptionally at least another fraction containing additional compounds(such as pesticides or plant growth regulators), which may for instancebe non-polar, may be separately recovered. Strobilurins, such astrifloxystrobin are examples of such additional compounds. The fractionscontaining cannabinoid compounds may be for instance one fractioncontaining Δ⁹-THC on the one hand and another fraction containing CBDand CBN on the other hand; or one fraction containing Δ⁹-THC, onefraction containing CBD, and one fraction containing CBN. Each of theabove fractions may be substantially free from the compounds indicatedas being contained in another fraction. Further additional compounds mayremain bound to the main stationary phase and may be released andcollected in a fraction during a subsequent washing step.

In other exemplary embodiments, some or all of the additional compoundsare eluted during the elution step, while the at least one cannabinoidcompound remains bound to the main stationary phase. The at least onecannabinoid compound can then be collected during a subsequent washingstep, as a fraction substantially free from the additional compoundseluted during the elution step.

The elution solution may be a non-polar solvent. The elution solutioncan also, more preferably, be a mixture of a polar and a non-polarsolvent.

By “polar solvent” is herein meant a solvent having a dielectricconstant at a temperature of 20° C. which is at least 15. By “non polarsolvent” is herein meant a solvent having a dielectric constant at atemperature of 20° C. which is less than 15.

The polar solvent can in particular be an alcohol, such as ethanol ormethanol, water and mixtures thereof. Preferably, ethanol is used as apolar solvent.

The non-polar solvent can in particular be an alkane, preferably a C5 toC10 alkane. More preferably, the non-polar solvent can be selected frompentane, hexane, heptane and octane. According to preferred embodiments,the non-polar solvent is hexane or heptane, more preferably heptane.

The elution may be performed in isocratic mode or gradient mode. In theisocratic mode, the composition of the elution solution remains constantduring the elution step. In the gradient mode, the composition of theelution solution varies during the elution step. In particular, thevolume proportion of polar solvent to non-polar solvent in the elutionsolution may vary during the elution step. In particular, the volumeproportion of polar solvent to non-polar solvent in the elution solutionmay increase during the elution step. The isocratic mode is howeverpreferred.

The elution solution can have a volume ratio of non-polar solvent topolar solvent from 99/1 to 50/50. More preferably, this volume ratio isfrom 98/2 to 60/40. Therefore, the volume ratio of non-polar solvent topolar solvent of the elution solution can be for instance from 99/1 to90/10; or from 90/10 to 80/20; or from 80/20 to 70/30; or from 70/30 to60/40; or from 60/40 to 50/50.

In the optional washing step, the main stationary phase can be washedwith a washing solution. During this step, compounds retained on themain stationary phase can be washed and optionally recovered, as one ormore fractions.

The washing solution may be a polar solvent. The washing solution canalso, more preferably, be a mixture of a polar and a non-polar solvent.

The polar solvent can in particular be an alcohol such as ethanol ormethanol, water and mixtures thereof. Preferably, ethanol is used as apolar solvent.

The non-polar solvent in particular be an alkane, preferably a C5 to C10alkane. More preferably, the non-polar solvent can be selected frompentane, hexane, heptane and octane. According to preferred embodiments,the non-polar solvent is hexane or heptane, heptane being morepreferred.

The washing solution can notably have a volume ratio of non-polarsolvent to polar solvent from 1/99 to 50/50. More preferably, thisvolume ratio is from 2/98 to 40/60. Therefore, the volume ratio ofnon-polar solvent to polar solvent of the washing solution can be from1/99 to 10/90; or from 10/90 to 20/80; or from 20/80 to 30/70; or from30/70 to 40/60; or from 40/60 to 50/50.

Preferably, the washing solution is more polar (i.e. contains a largerrelative proportion of polar solvent) than the elution solution.

The washing may be performed in isocratic mode or gradient mode. In theisocratic mode, the composition of the washing solution remains constantduring the washing step. In the gradient mode, the composition of thewashing solution varies during the washing step. In particular, thevolume proportion of polar solvent to non-polar solvent in the washingsolution may vary during the washing step. In particular, the volumeproportion of polar solvent to non-polar solvent in the washing solutionmay increase during the washing step. The isocratic mode is howeverpreferred.

Optionally, the main stationary phase can be equilibrated by putting itin contact with an equilibration solution. This step makes theadsorption sites of the main stationary phase available for anotherpurification.

The equilibration solution may be a non-polar solvent. The equilibrationsolution can also, more preferably, be a mixture of a polar and anon-polar solvent.

The polar solvent can in particular be an alcohol such as ethanol ormethanol, water and mixtures thereof. Preferably, ethanol is used as apolar solvent.

The non-polar solvent can in particular be an alkane, preferably a C5 toC10 alkane. More preferably, the non-polar solvent can be selected frompentane, hexane, heptane and octane. According to preferred embodiments,the non-polar solvent is hexane or heptane, heptane being morepreferred.

The equilibration solution can notably have a volume ratio of non-polarsolvent to polar solvent from 99/1 to 50/50. More preferably, thisvolume ratio is from 98/2 to 60/40. Therefore, the volume ratio ofnon-polar solvent to polar solvent of the equilibration solution can befrom 99/1 to 90/10; or from 90/10 to 80/20; or from 80/20 to 70/30; orfrom 70/30 to 60/40; or from 60/40 to 50/50.

Preferably, the equilibration solution is the same as the elutionsolution.

Preliminary Purification Stage by Chromatographic Purification

The method of the invention may comprise two successive stages, apreliminary purification stage or pretreatment (described below) and amain purification stage (described above). In the preliminary stage, theinitial mixture is prepared from a preliminary mixture corresponding tothe starting material described above. In this stage, use is made of apreliminary stationary phase.

The preliminary purification stage may be a stage of chromatographicpurification.

The preliminary stationary phase may be the same or different from themain stationary phase.

According to some embodiments, the preliminary stationary phase maycomprise silica particles. In particular, the preliminary stationaryphrase may comprise or consist of silica particles comprising aminoand/or diol groups, as described above.

If the preliminary and main stationary phases are the same, use may bemade of two different beds of the same stationary phase, for instance intwo different chromatographic installations. Alternatively, thepreliminary and main stationary phases may be the same bed in the samechromatographic installation, successively used at different points intime.

According to some embodiments, the preliminary stationary phase maycomprise or consist of non-functionalized silica particles.

During a first step, the preliminary mixture is put in contact with thepreliminary stationary phase by injecting the preliminary mixture ontothe preliminary stationary phase.

An elution is then carried out with an elution solution in order tocollect one or more preliminary elution fractions, optionally followedby a washing step with a washing solution in order to collect one ormore preliminary washing fractions.

At least one preliminary elution fraction is collected at the elutionstep.

If the washing step is carried out, at least one preliminary washingfraction is collected at the washing step.

In some embodiments, a single preliminary elution fraction is collectedat the elution step. In other embodiments, at least two preliminaryelution fractions are collected at the elution step.

In some embodiments, a single preliminary washing fraction is collectedat the washing step. In other embodiments, at least two preliminarywashing fractions are collected at the washing step.

More than one preliminary fraction may be collected at different pointsin time in a given step.

At least one of the preliminary elution fractions or the preliminarywashing fractions is a liquid fraction enriched in the at least onecannabinoid compound and the one or more additional compounds anddepleted in the further compounds. Said liquid fraction provides theinitial mixture and is used in the main purification stage.

Each elution step, each elution solution, each washing step and eachwashing solution may be as described in more detail above with respectto the main purification stage. The elution solution of the preliminarypurification stage may be the same as the elution solution of the mainpurification stage, or preferably different.

In some embodiments, the elution solution of the main purification stageis more polar than the elution solution of the preliminary purificationstage.

In some embodiments, the elution solution of the main purification stageis less polar than the elution solution of the preliminary purificationstage.

The elution solution of the preliminary purification stage is preferablya mixture of a polar and a non-polar solvent.

The polar solvent can in particular be an alcohol, such as ethanol ormethanol, water and mixtures thereof. Preferably, ethanol is used as apolar solvent.

The non-polar solvent can in particular be an alkane, preferably a C5 toC10 alkane. More preferably, the non-polar solvent can be selected frompentane, hexane, heptane and octane. According to preferred embodiments,the non-polar solvent is hexane or heptane, more preferably heptane.

The elution solution of the preliminary purification stage can have avolume ratio of non-polar solvent to polar solvent from 99/1 to 50/50.More preferably, this volume ratio is from 98/2 to 60/40. Therefore, thevolume ratio of non-polar solvent to polar solvent of the elutionsolution of the preliminary purification stage can be for instance from99/1 to 90/10; or from 90/10 to 80/20; or from 80/20 to 70/30; or from70/30 to 60/40; or from 60/40 to 50/50.

The preliminary stationary phase may be equilibrated after the elutionand optional washing of the preliminary purification stage, as describedabove with respect to the main purification stage. This is useful inparticular if it is to be used again as the main stationary phase at themain purification stage.

This variant may be advantageous in order to separate the at least onecannabinoid compound from several different compounds or groups ofcompounds. At the preliminary purification stage, the at least onecannabinoid compound is separated from the further compounds but issubstantially not separated from the additional compounds.

As a result, the initial mixture provided by the preliminarypurification stage is enriched in the at least one cannabinoid compoundand the additional compounds and depleted in the further compounds,relative to the preliminary mixture.

The ratio of the weight concentration of the at least one cannabinoidcompound relative to the weight concentration of the further compoundsis greater in the initial mixture than in the preliminary mixture,preferably by at least a factor of 10 or 100 or 1000 or 10000.

The weight proportion of the at least one cannabinoid compound in theinitial mixture relative to the amount of the at least one cannabinoidcompound present in the preliminary mixture may be at least 95%,preferably at least 98%, more preferably at least 99%, and mostpreferably at least 99.5% or 99.9%.

The weight proportion of the one or more further compounds recovered inthe initial mixture relative to the amount present in the preliminarymixture may be less than 5%, preferably at least 2%, more preferablyless than 1%, and most preferably less than 0.5% or less than 0.1%.

The weight proportion of the one or more further compounds relative tothe at least one cannabinoid compound in the initial mixture may inparticular be less than 2%, preferably less than 1%, more preferablyless than 0.5%, most preferably less than 0.1% or less than 0.05% orless than 0.01%.

Then, at the main purification stage, the at least one cannabinoidcompound is separated from the additional compounds. As a result, at themain purification stage, a fraction is collected which is enriched inthe at least one cannabinoid compound and depleted in the additionalcompounds (relative to the initial mixture obtained after thepreliminary purification stage).

The main benefits of a two-stage process are that the size of thecolumns and volumes of solvents can be adjusted to the specificseparation of the at least one cannabinoid compound from the furthercompounds at the preliminary purification stage and to the specificseparation of the at least one cannabinoid compound from the additionalcompounds at the main purification stage.

In preferred embodiments, the preliminary purification stage is designedto separate the at least one cannabinoid compound from strongly retainedimpurities (further compounds). The retention time differences beinghigher during the preliminary purification stage, a reduced amount ofstationary phase and of solvents is required, compared to the mainpurification stage.

In some embodiments, the additional compounds may comprise non-polarplant growth regulators and/or pesticides (such as in particularstrobilurins such as trifloxystrobin) and optionally cannabinoidcompounds; and the further compounds may comprise polar plant growthregulators and/or pesticides (such as in particular neonicotinoids, e.g.imidacloprid and thiamethoxam and/or daminozide).

In the main purification stage, one particular (desired) cannabinoidcompound, for instance selected from the group of CBN, CBD and Δ⁹-THC,may be separated from one or more other cannabinoid compounds, forinstance also selected from the group of CBN, CBD and Δ⁹-THC, and mayalso be separated from non-polar plant growth regulators and/orpesticides (such as a strobilurin, e.g. trifloxystrobin). Severaldistinct fractions can thus be collected in the main purification stage.

Preliminary Purification Stage by Suspension Purification

As a variant to the above preliminary purification stage bychromatographic purification, the preliminary purification stage mayalso be conducted by putting the preliminary mixture in contact with thepreliminary stationary phase, in a suspension solution to form asuspension. For example, the preliminary stationary phase may be mixedwith the preliminary mixture (and the suspension solution), in a tankprovided with stirring elements. Such stirring elements can for exampleinclude one or more agitators, a recirculation pump or the like. Thetank may be for example a fluidized bed reactor. After a given residencetime, the suspension can be filtered, collecting a first fraction on theone hand and the preliminary stationary phase on the other hand.

Optionally, the preliminary stationary phase may be resuspended in afurther solution, and filtered so as to collect a further fraction. Thisstep can be repeated several times if needed.

At least one of the first or the further fractions is a liquid fractionenriched in the at least one cannabinoid compound and the one or moreadditional compounds and depleted in the further compounds. Said liquidfraction provides the initial mixture and is used in the mainpurification stage.

The preliminary stationary phase used in the suspension purification maybe the same as the preliminary stationary phase used in the preliminarychromatographic purification, as described above.

Alternatively, the preliminary stationary phase may comprise or consistof activated charcoal.

Alternatively, the preliminary stationary phase may comprise or consistof one or more zeolites.

The suspension solution may be an elution solution as described above inrelation with the preliminary purification stage by chromatographicpurification.

According to some embodiments, the further solution used for theresuspension of the preliminary stationary phase can also be an elutionsolution as described above in relation with the preliminarypurification stage by chromatographic purification.

According to some embodiments, the further solution used for theresuspension of the preliminary stationary phase can be a washingsolution as described above in relation with the preliminarypurification stage by chromatographic purification.

According to some embodiments, and in case the preliminary stationaryphase is resuspended more than once, for example two times, the firstresuspension can be carried out with an elution solution as describedabove in relation with the preliminary purification stage bychromatographic purification, and the second resuspension can be carriedout with a washing solution as described above in relation with thepreliminary purification stage by chromatographic purification.

Optionally, the preliminary stationary phase can finally be suspended inan equilibration solution as described above in relation with thepreliminary purification stage by chromatographic purification toachieve the equilibration of the preliminary stationary phase beforereusing the preliminary stationary phase for another purification.

Chromatographic Installations and Operating Conditions

The method according to the invention may be discontinuous (or batch),semi-continuous or continuous.

The method according to the invention can be carried out at a constanttemperature or at different successive temperatures. Preferably thetemperature is substantially constant. The temperature at which themethod is carried out may preferably range from 10 to 40° C., morepreferably from 15 to 30° C. and most preferably from 20 to 25° C.

The term “chromatographic purification stages” used below, refers to themain purification stage as well as the preliminary purification stagewhen carried out by a chromatographic purification.

The chromatographic purification stages according to the invention arepreferably carried out at a pressure from 1 to 100 bar and preferablyfrom 10 to 40 bar.

The chromatographic purification stages according to the invention maybe implemented in a chromatographic installation having a static bed orpreferably in a chromatographic installation with a non-static bed.

In a chromatographic installation with a static bed, the mixture ofcompounds to be separated percolates in a generally cylindricalenclosure (or column). The column contains a bed of porous material(stationary phase) which is permeable to fluids. The percolation rate ofeach compound in the mixture depends on the physical properties of thecompound. The most retained compounds on the stationary phase percolatemore slowly than the less retained compounds on the stationary phase.

It is possible to carry out such a treatment in several columns inseries or in parallel, but generally a chromatographic separationinstallation system with a static bed is applied with a single column.

Examples of such chromatographic installations with a static bed are theHPLC (High Performance Liquid Chromatography) or CYCLOJET™ (a systemwith recycling in the stationary state) systems.

The CYCLOJET™ system is as described in document U.S. Pat. No.6,063,284, to which reference is expressly made. This is a discontinuouschromatographic separation installation with a single column, in whichthe most retained species (i) and then the less retained species (ii)are collected separately at the outlet of the column, the non-separatedportion of the chromatogram being recycled by the main pump, and themixture to be separated being periodically injected by means of aninjection loop essentially located in the middle of the recycled portionof the chromatogram. After several chromatographic cycles, the processreaches a periodical stationary state in which the amount of injectedproducts is equal to the amount of separated products collectedseparately at the outlet of the column.

An alternative of the CYCLOJET™ system using two columns is described indocument U.S. Pat. No. 5,630,943, to which reference is expressly made.

An installation with a non-static bed is a multi-column installation, inwhich the relative positions of the stationary phase bed and of theinjection or collecting points of the flows move over time.

Examples of such chromatographic installations with a non-static bed arethe SMB (simulated moving bed), iSMB (improved simulated moving bed),SSMB (sequential simulated moving bed), AMB (actual moving bed),VARICOL™, MODICON™ POWERFEED™, MCSGP or GSSR (multi column gradientchromatography) systems.

An SMB system comprises a plurality of individual columns containing anadsorbent, which are connected in series. An eluent flow crosses thecolumns along a first direction. The injection points of the feed flowand of the eluent flow, as well as the points for collecting theseparated compounds, are periodically and simultaneously shifted bymeans of a set of valves. The global effect is to simulate the operationof a single column containing a moving bed of solid adsorbent, the solidadsorbent moving in a counter current direction relatively to the eluentflow. Thus, an SMB system consists of columns which contain stationarybeds of solid adsorbent through which the eluent passes, but theoperation is such that a continuous moving bed as a counter current issimulated.

The most conventional form of an SMB system is the SMB system with fourzones. Other possible forms are the SMB systems with three zones and theSMB systems with two zones (as described in the article “Two SectionSimulated Moving Bed Process” of Kwangnam Lee, in Separation Science andTechnology 35(4):519-534, 2000, to which reference is expressly made).

In the iSMB and SSMB systems, there is at least one step in which thesystem operates in a closed loop, without any product entry or exit.

An iSMB system is as described in documents EP 0 342 629 and U.S. Pat.No. 5,064,539, to which reference is expressly made.

An SSMB system divides the introductions and collections of the flowsinto subsequences applied periodically.

Other alternatives of the SMB systems are: the SMB system varying overtime and the POWERFEED™ system, as described in document U.S. Pat. No.5,102,553 and in the article “PowerFeed operation of simulated movingbed units: changing flow-rates during the switching interval”, of Zhanget al. in Journal of Chromatography A, 1006:87-99, 2003, to whichreference is expressly made; the MODICON™ system as described indocument U.S. Pat. No. 7,479,228, to which is reference is expresslymade; and the SMB system with internal recirculation, as described indocument U.S. Pat. No. 8,282,831, to which reference is expressly made.

An AMB system has an operation similar to an SMB system. However,instead of displacing the feed flow and eluent injection points, as wellas the collecting points, by means of a set of valves, a set ofadsorption units (columns) are physically displaced relatively to thefeed and collecting points. Again, the operation allows simulation of acontinuous counter current moving bed.

A VARICOL™ chromatography system is as described in documents U.S. Pat.Nos. 6,136,198, 6,375,839 6,413,419 and 6,712,973, to which reference isexpressly made. A VARICOL™ system comprises a plurality of individualcolumns containing an adsorbent, which are connected in series. Aneluent is passed into the columns along a first direction. The injectionpoints for the mixture to be separated and for the eluent and the pointsfor collecting the separated compounds in the system are periodicallydisplaced asynchronously, by means of a set of valves. The global effectis to generate separation zones with a variable length over time,thereby allocating the stationary phase dynamically in the zones whereit is the most useful, and allowing a similar power of separation withless chromatographic separation units and increased productivity.

The chromatographic purification stages of the present invention may becarried out in a single-column chromatographic installation.

The chromatographic purification stages of the present invention mayalso be carried out in a multicolumn chromatographic installation with adiscontinuous feed injection, such as but not limited to BioSC (asdescribed in document EP 2040811) or DCC processing (as described indocument EP 1912716).

The chromatographic purification stages of the present invention arepreferably carried out in a high-pressure liquid chromatographyinstallation.

According to some embodiments, the method of the invention onlycomprises the main purification stage (without the preliminarypurification stage) and it can be performed in a single columninstallation, or in a two or three-column installation chosen betweenMCSGP or GSSR or DCC, or in a four or five or six or seven oreight-column installation chosen between SMB, iSMB, SSMB, AMB, VARICOL™,MODICON™ and POWERFEED™ In all of the above installations, the total bedlength for the stationary phase preferably ranges from 10 cm to 100 cm.

According to some embodiments, the method of the present inventioncomprises the preliminary purification stage and the main purificationstage and:

The preliminary purification stage may be carried out by using a singlecolumn installation, or by using a two or three-column installation suchas DCC, or by using a four or five or six or seven or eight-columninstallation chosen between BioSC, SMB, iSMB, SSMB, AMB, VARICOL™,MODICON™ and POWERFEED™. In all of the above installations, the totalbed length for the stationary phase preferably ranges from 10 cm to 100cm.

The main purification stage may be carried out by using a single columninstallation, or by using a two or three-column installation chosenbetween MCSGP or GSSR or DCC, or by using a four or five or six or sevenor eight-column installation chosen between SMB, iSMB, SSMB, AMB,VARICOL™, MODICON™ and POWERFEED™ In all of the above installations, thetotal bed length for the stationary phase preferably ranges from 10 cmto 100 cm.

The chromatographic purification stages of the present invention may becarried out in one or more installations comprising one or morechromatographic columns having a diameter (internal diameter) equal toor larger than 5 cm. For example, the diameter of the one or morechromatographic columns can be from 5 to 10 cm; or from 10 to 20 cm; orfrom 20 to 30 cm; or from 30 to 40 cm; or from 40 to 50 cm; or from 50to 100 cm; or from 100 to 150 cm; or from 150 to 200 cm.

The chromatographic installation(s) used in the present invention mayhave a total dead volume lower than or equal to 30%, preferably lowerthan or equal to 20%, more preferably lower than or equal to 10% andeven more preferably lower than or equal to 5% of the total volume of(main and/or preliminary) stationary phase in the installation (i.e. thevolume of the bed of stationary phase).

The term “dead volume” can be defined as the sum of:

-   -   the volumes of piping in the chromatographic installation        between a valve of the inlet for the injection of the feed        (initial mixture or preliminary mixture) and the inlet of the        bed of the stationary phase;    -   the volumes of piping in the chromatographic installation        between an outlet of the bed of the stationary phase and a valve        of the outlet for the collection of the product of interest;    -   the volumes of piping in the chromatographic installation        connecting any column in series between a valve of the outlet        for the collection of the product of interest and a valve of the        inlet for the injection of the feed (initial mixture or        preliminary mixture), if the installation is multicolumn.

The velocity of the fluids at the entrance of the bed of (main and/orpreliminary) stationary phase can be from 30 cm/hour to 5000 cm/hour,preferably from 100 to 4000 cm/hour, and more preferably from 200 to3000 cm/hour. For example, this velocity can be from 30 to 40 cm/hour;or from 40 to 50 cm/hour; or from 50 to 60 cm/hour; or from 60 to 70cm/hour; or from 70 to 80 cm/hour; or from 80 to 90 cm/hour; or from 90to 100 cm/hour; or from 100 to 250 cm/hour; or from 250 to 500 cm/hour;or from 500 to 750 cm/hour; or from 750 to 1000 cm/hour; or from 1000 to1500 cm/hour; or from 1500 to 2000 cm/hour; or from 2000 to 3000cm/hour; or from 3000 to 4000 cm/hour; or from 4000 to 5000 cm/hour.

These ranges may apply to the initial mixture, and/or the preliminarymixture, and/or the elution solution, and/or the washing solution,and/or the equilibration solution.

The velocity of the liquid can be constant over the various steps of onechromatographic stage (either the main purification stage or thepreliminary purification stage carried out by chromatographicpurification).

Alternatively, the velocity of the liquid can vary during onechromatographic stage. For example, the velocity of the initial mixtureor the preliminary mixture can be lower than the velocity of the elutionsolution, and/or the velocity of the initial mixture or the preliminarymixture can be lower than the velocity of the washing solution, and/orthe velocity of the initial mixture or the preliminary mixture can belower than the velocity of the equilibration solution. Otherwise, thevelocity of the initial mixture or the preliminary mixture can be higherthan the velocity of the elution solution, and/or the velocity of theinitial mixture or the preliminary mixture can be higher than thevelocity of the washing solution, and/or the velocity of the initialmixture or the preliminary mixture can be higher than the velocity ofthe equilibration solution.

Similarly, the velocity of the elution solution, the washing solutionand the equilibration solution may be different, lower or higher fromone another.

EXAMPLES

The following examples illustrate the invention without limiting it.

The starting material to be purified comprises Δ⁹-tetrahydrocannabinol,cannabidiol, cannabinol, daminozide, imidacloprid, thiamethoxam andtrifloxystrobin.

Example 1—Chromatographic Separation of Imidacloprid and Thiamethoxam

In this example, the invention is applied on a HPLC Agilent 1100.

The stationary phase used is Amino 10 μm, M.S. Gel and a mixture ofethanol and heptane in a volume ratio of 60/40 heptane/ethanol is usedas the mobile phase.

As shown in FIG. 1, the cannabinoid compounds Δ⁹-THC, CBD and CBN (A)co-elute with trifloxystrobin (B), while imidacloprid (C) andthiamethoxam (D) are successfully separated. Furthermore, daminozide(not shown in FIG. 1) remains retained in the stationary phase.

Example 2—Chromatographic Separation of Daminozide, Imidacloprid andThiamethoxam

This example is similar to example 1, expect that the stationary phaseused is Daisogel Diol 10 μm. A mixture of ethanol and heptane in avolume ratio of 60/40 heptane/ethanol is still used as the mobile phase.

As shown in FIG. 2, the cannabinoid compounds Δ⁹-THC, CBD, and CBN (A)co-elute with trifloxystrobin (B), while daminozide (C), imidacloprid(D) and thiamethoxam (E) are successfully separated.

Example 3—Chromatographic Separation of Trifloxystrobin and Δ⁹-THC

This example is similar to example 1, expect that a less polar mixtureof ethanol and heptane in a volume ratio of 98/2 heptane/ethanol is usedas the mobile phase.

As shown in FIG. 3, trifloxystrobin (A) and Δ⁹-THC (B) no longerco-elute, while CBD, and CBN (C) still elute together. Furthermore,daminozide, imidacloprid, and thiamethoxam (not shown in FIG. 3) remainretained on the stationary phase.

Example 4—Chromatographic Separation of Trifloxystrobin, Δ⁹-THC, CBN andCBD

This example is similar to example 2, expect that a mixture of ethanoland heptane in a volume ratio of 98/2 heptane/ethanol is used as themobile phase.

As shown in FIG. 4, trifloxystrobin, (A), Δ⁹-THC (B), CBN (C) and CBD(D) are successfully separated. Daminozide, imidacloprid, andthiamethoxam (not shown in FIG. 3) remain retained on the stationaryphase.

Example 5—Chromatographic Separation of Trifloxystrobin, Δ⁹-THC, CBN,CBD, Daminozide, Imidacloprid and Thiamethoxam

In this example, the invention is implemented on a High-Pressure LiquidChromatography installation.

The stationary phase used is Daisogel Diol 10 μm.

Firstly, the stationary phase is eluted with a mixture of ethanol andheptane in a volume ratio of 98/2 heptane/ethanol and as shown in FIG.5. Within the first 13 minutes, trifloxystrobin (A),Δ⁹-tetrahydrocannabinol (B), cannabinol (C), and cannabidiol (D) arepurified.

Then washing is performed with a more polar mixture of ethanol andheptane (volume ratio of 20/80 heptane/ethanol) in order to collect themore polar daminozide (E), imidacloprid (F), and thiamethoxam (G).

Finally, a mixture of ethanol and heptane in a volume ratio of 98/2heptane/ethanol is used for the equilibration of the stationary phase.

Example 6—Two Step Chromatographic Separation of Trifloxystrobin, THC,CBN, CBD, Daminozide, Imidacloprid and Thiamethoxam

In this example, the invention is applied on a HPLC Agilent 1100.

The stationary phase used is Daisogel Diol 10 μm.

In a first step, the stationary phase is eluted with a mixture ofethanol and heptane in a volume ratio of 60/40 heptane/ethanol and theso-called initial mixture is collected on the one hand, comprisingΔ⁹-THC, CBN and CBD, while the more polar daminozide, imidacloprid, andthiamethoxam are successfully separated on the other hand.

In a second step, the initial mixture is loaded in a HPLC Agilent 1100and the stationary phase is eluted with a mixture of ethanol and heptanein a volume ratio of 98/2 heptane/ethanol. At the end of this step,trifloxystrobin, Δ⁹-THC, CBN, and CBD are successfully purified.

These examples demonstrate that the separation of at least onecannabinoid compound from a starting material is possible and that thevalues of retention time can vary from one example to another or fromone stationary phase to another. In fact, the difference in retentiontimes which is observed is sufficient in order to purify the startingmaterial at an industrial scale. From those examples, and using achromatography simulation software, based for example on methodsdescribed in the Preparative Chromatography of Fine Chemicals andPharmaceutical Agents, Henner Schmidt-Traub, Wiley-VCH, ISBN-13978-3-527-30643-5, it is possible to produce a model from anychromatogram that presents a difference in retention times. This modelmakes it possible to predict that a purity higher than 99%, and a yieldgreater than 90% for the various tested cannabinoid compounds, can beachieved, for any initial weight proportions of cannabinoids andadditional compounds in the starting material.

1. A method for the chromatographic purification of at least onecannabinoid compound, wherein the method comprises a main purificationstage comprising the steps of: injecting an initial mixture comprisingthe at least one cannabinoid compound and one or more additionalcompounds onto a main stationary phase comprising silica particles, thesilica particles comprising amino and/or diol groups; performing anelution with an elution solution, and collecting one or more elutionfractions; and optionally, washing the main stationary phase with awashing solution and collecting one or more washing fractions; at leastone of the elution fractions or washing fractions containing the atleast one cannabinoid compound purified from the one or more additionalcompounds.
 2. The method according to claim 1, further comprising apreliminary purification stage before the main purification stage, saidpreliminary purification stage comprising the steps of: putting apreliminary mixture comprising the at least one cannabinoid compound,the one or more additional compounds and one or more further compoundsin contact with a preliminary stationary phase; and collecting at leastone liquid fraction enriched in the at least one cannabinoid compoundand the one or more additional compounds and depleted in the furthercompounds, said at least one liquid fraction providing the initialmixture.
 3. The method according to claim 2, wherein the preliminarystationary phase comprises silica particles comprising amino and/or diolgroups.
 4. The method according to claim 2 wherein the preliminarypurification stage comprises putting in contact the preliminary mixturewith the preliminary stationary phase by injecting the preliminarymixture onto the preliminary stationary phase; performing an elutionwith an elution solution, and collecting one or more preliminary elutionfractions; and optionally washing the preliminary stationary phase witha washing solution and collecting one or more preliminary washingfractions; at least one of said preliminary elution fractions and/orpreliminary washing fractions being the liquid fraction enriched in theat least one cannabinoid compound and the one or more additionalcompounds and depleted in the further compounds.
 5. The method accordingto claim 1, wherein the initial mixture is a Cannabis extract.
 6. Themethod according to claim 1, wherein the at least one cannabinoidcompound is selected from Δ⁹-tetrahydrocannabinol, cannabidiol,cannabinol, cannabigerol, cannabichromene, cannabidivarol,tetrahydrocannabidiol, tetrahydrocannabidivarol,Δ⁸-tetrahydrocannabinol, carboxylic acid precursors of the foregoingcompounds, related naturally occurring compounds and their derivatives,and combinations thereof.
 7. The method according to claim 1, whereinthe one or more additional compounds are selected from cannabinoidcompounds, pesticides, plant growth regulators and combinations thereof.8. The method according to claim 2, wherein the one or more furthercompounds are selected from cannabinoid compounds, pesticides, plantgrowth regulators and combinations thereof.
 9. The method according toclaim 1, wherein each of the elution solution and the washing solutionis a mixture of a polar solvent and a non-polar solvent; the polarsolvent independently being selected from ethanol, methanol and water,and the non-polar solvent being independently selected from pentane,hexane, heptane and octane.
 10. The method according to claim 1, whereineach of the elution solution and the washing solution is a mixture of apolar solvent and a non-polar solvent; the elution solution having avolume ratio of non-polar solvent to polar solvent from 99/1 to 50/50;and the washing solution having a volume ratio of non-polar solvent topolar solvent from 1/99 to 50/50.
 11. The method according to claim 1,further comprising equilibrating the main stationary phase with anequilibration solution in order to equilibrate the main stationary phasefor a further injection, the elution solution and the equilibrationsolution being the same.
 12. The method according to claim 1, which iscarried out in an installation chosen from a single column installation,or a multi-column installation having a total bed length smaller than100 cm.
 13. The method according to claim 2, wherein the preliminarypurification stage is carried out in an installation chosen from asingle column installation, or a multi-column installation having atotal bed length smaller than 100 cm, and the main purification stage iscarried out in a multi-column installation having a total bed lengthsmaller than 100 cm.
 14. The method according to claim 1, which iscarried out in an installation comprising one or more chromatographiccolumns having a diameter equal to or larger than 5 cm.
 15. The methodaccording to claim 14, wherein the installation has a total dead volumelower than or equal to 30% of the total volume of main stationary phasein the installation.
 16. The method according to claim 1, wherein theinjection of the initial mixture is performed continuously.
 17. Themethod according to claim 1, wherein the injection of the initialmixture is performed in a discontinuous way.
 18. The method according toclaim 1, wherein the weight proportion of the purified at least onecannabinoid compound relative to the amount of the at least onecannabinoid compound present in the initial mixture is at least 95%. 19.The method according to claim 1, wherein the at least one elutionfraction or washing fraction containing the at least one cannabinoidcompound purified from the one or more additional compounds furthercomprises a weight proportion of the one or more additional compoundswhich is less than 5%, relative to the amount of the one or moreadditional compounds present in the initial mixture.
 20. The methodaccording to claim 2 wherein the preliminary purification stagecomprises putting the preliminary mixture in contact with thepreliminary stationary phase in a suspension solution, to form asuspension; filtering so as to collect a first fraction; optionally,resuspending the preliminary stationary phase in one or more furthersolutions, and filtering so as to collect one or more further fractions;the liquid fraction enriched in the at least one cannabinoid compoundand the one or more additional compounds and depleted in the furthercompounds being at least one of the first and further fractions.